Active efflux and diffusion are involved in transport of Pseudomonas aeruginosa cell-to-cell signals.

1Department of Microbiology and Immunology, University of Rochester, Rochester, New York 14642, USA.

Abstract

Many gram-negative bacteria communicate by N-acyl homoserine lactone signals called autoinducers (AIs). In Pseudomonas aeruginosa, cell-to-cell signaling controls expression of extracellular virulence factors, the type II secretion apparatus, a stationary-phase sigma factor (sigmas), and biofilm differentiation. The fact that a similar signal, N-(3-oxohexanoyl) homoserine lactone, freely diffuses through Vibrio fischeri and Escherichia coli cells has led to the assumption that all AIs are freely diffusible. In this work, transport of the two P. aeruginosa AIs, N-(3-oxododecanoyl) homoserine lactone (3OC12-HSL) (formerly called PAI-1) and N-butyryl homoserine lactone (C4-HSL) (formerly called PAI-2), was studied by using tritium-labeled signals. When [3H]C4-HSL was added to cell suspensions of P. aeruginosa, the cellular concentration reached a steady state in less than 30 s and was nearly equal to the external concentration, as expected for a freely diffusible compound. In contrast, [3H]3OC12-HSL required about 5 min to reach a steady state, and the cellular concentration was 3 times higher than the external level. Addition of inhibitors of the cytoplasmic membrane proton gradient, such as azide, led to a strong increase in cellular accumulation of [3H]3OC12-HSL, suggesting the involvement of active efflux. A defined mutant lacking the mexA-mexB-oprM-encoded active-efflux pump accumulated [3H]3OC12-HSL to levels similar to those in the azide-treated wild-type cells. Efflux experiments confirmed these observations. Our results show that in contrast to the case for C4-HSL, P. aeruginosa cells are not freely permeable to 3OC12-HSL. Instead, the mexA-mexB-oprM-encoded efflux pump is involved in active efflux of 3OC12-HSL. Apparently the length and/or degree of substitution of the N-acyl side chain determines whether an AI is freely diffusible or is subject to active efflux by P. aeruginosa.

Accumulation of radiolabeled compounds by P. aeruginosa. (A) [14C]leucine accumulation in strain PAO1 treated with chloramphenicol (5 mM); (B) accumulation of [3H]3OC12-HSL (circles) and [3H]C4-HSL (triangles) in strain PAO1; (C) accumulation of [3H]3OC12-HSL (circles) and [3H]C4-HSL (triangles) in mexAB-orpM mutant strain PAO200. Closed symbols, non-azide-treated cells; open symbols, cells treated with sodium azide (30 mM). Accumulation of each compound was determined as described in Materials and Methods. Data are the averages (± SDs) of results from two to five independent experiments.

Efflux of [3H]C4-HSL (A) and [3H]3OC12-HSL (B) from P. aeruginosa PAO1 (wild type), PAO-JP3 (lasR rhlR), or PAO200 (mexABA-oprM). Open bars, radioactivity loaded in cells; closed bars, radioactivity remaining in cells after suspension in AI-free buffer. Cells were de-energized with CCCP (250 μM) as indicated. Data are the averages (± SDs) of results from two to four independent experiments.